Regulation of EMT During Avian Heart Valve Formation

描述（由申请人提供）：在心脏发育过程中，由于TGF¿介导的上皮-间充质细胞转化（EMT），瓣膜祖细胞首先出现在房室管中。以往的研究表明，这种EMT可分为两个阶段，即激活阶段和侵袭阶段。第1阶段部分由转化生长因子β 2 （TGF¿2）介导，第2阶段部分由TGF¿3介导。先前的研究表明，EMT可以通过抑制5种不同的TGF -受体、TGF -受体II、TGF -受体III、内啡肽、ALK2和ALK5而被破坏。这是我们的假设，有两个离散的信号复合物介导TGF -调控的EMT。为了继续分析TGF¿介导的正常心脏发育调节，第一个目的是研究特定TGF¿亚型和受体在介导EMT调节中的相关性。微阵列实验将评估特定TGF¿亚型的缺失对EMT前组织、EMT期间和EMT后间充质细胞中基因表达改变的影响。TGF -调控基因中选定的标记物将被用来评估当每个特定受体被破坏时，基因表达是否会改变。这些数据之间的相关性将指向介导EMT的特定受体和配体信号复合物。这些假定的复合物将在蛋白质水平上得到证实。来自其他实验室的数据表明，心脏EMT中存在多种信号转导机制和细胞内调节因子，包括ErbB2、NF-1、VEGF和NFATc1。在第二个目标中，将进行实验来确定TGF¿信号的破坏是否会改变任何其他候选机制，或者这些分子的丢失是否会改变TGF¿配体或受体的表达。在神经嵴系统中观察到，在神经管中强制表达sox8、9或10会引起EMT并产生异位的神经嵴细胞。我们推测这类似于心脏中的细胞入侵步骤，并且需要一些TGF - γ活性。在第三个目的中，我们将进行实验，以确定外源的Sox基因表达是否会强制EMT，以及Sox基因的表达是否受TGF¿信号转导的调节或调节TGF¿介导的反应。总之，这些实验将揭示EMT调节的新机制，并有助于了解先天性心脏病。

DESCRIPTION (provided by applicant): During cardiac development, valve progenitors first arise in the atrioventricular canal as a result of a TGF¿-mediated epithelial-mesenchymal cell transformation (EMT). Prior studies showed that this EMT can be divided into two stages, an activation stage and an invasion stage. Stage 1 is mediated, in part, by transforming growth factor beta 2 (TGF¿2) and stage 2 is mediated, in part, by TGF¿3. Previous work showed that EMT can be disrupted by the inhibition of 5 different TGF¿ receptors, TGF¿ receptor II, TGF¿ receptor III, Endoglin, ALK2 and ALK5. It is our hypothesis that there are two more discrete signaling complexes mediating TGF¿-regulated EMT. To continue the analysis of TGF¿-mediated regulation of normal heart development, the first aim will examine the correlation between specific TGF¿ isoforms and receptors in mediating EMT regulation. Microarray experiments will be performed to assess the effect loss of specific TGF¿ isoforms on altered gene expression in tissues prior to EMT, during EMT and in mesenchymal cells after EMT. Selected markers among the TGF¿-regulated genes will then be used to assess whether gene expression is altered as each of the specific receptors is disrupted. Correlations between these data will point to specific receptor and ligand signaling complexes that mediate EMT. These putative complexes will be confirmed at the protein level. Data from other labs has implicated a variety of signal transduction mechanisms and intracellular regulators in cardiac EMT including ErbB2, NF-1, VEGF and NFATc1. In the second aim, experiments will be undertaken to determine whether disruption of TGF¿ signaling alters any of the other candidate mechanisms or whether loss of these molecules alters TGF¿ ligand or receptor expression. It was observed in the neural crest system that forced expression of Sox 8, 9 or 10 in the neural tube would cause an EMT and produce ectopic neural crest cells. We conjecture that this is analogous to the cell invasion step in the heart and that it requires some TGF¿ activity. In the third aim, experiments will be undertaken to determine whether exogenous expression of Sox genes will force EMT and whether Sox gene expression is regulated by TGF¿ signal transduction or regulates TGF¿-mediated responses. Together these experiments will shed new light on the mechanisms of EMT regulation and aid in an understanding of congenital heart disease.